CN113604611B

CN113604611B - Digital PCR method for detecting B1 group adenovirus

Info

Publication number: CN113604611B
Application number: CN202110920004.1A
Authority: CN
Inventors: 陈威巍; 许文; 杨俊连; 杨欣欣; 徐哲; 涂波; 黄磊
Original assignee: Fifth Medical Center of PLA General Hospital
Current assignee: Fifth Medical Center of PLA General Hospital
Priority date: 2021-08-11
Filing date: 2021-08-11
Publication date: 2022-03-08
Anticipated expiration: 2041-08-11
Also published as: CN113604611A

Abstract

The invention provides a primer for detecting B1 group adenovirus, and a probe primer combination, a reagent and a kit comprising the primer. The detection method using the product has high accuracy, higher sensitivity than that of common PCR and quantitative detection.

Description

Digital PCR method for detecting B1 group adenovirus

Technical Field

The invention relates to the technical field of biology, in particular to a digital PCR method for detecting B1 group adenovirus.

Background

Currently, digital PCR includes both droplet-type PCR detection methods and chip-type detection methods. The number of effective reaction cavities on a single chip in the chip-based detection method is generally only thousands, which is far less than that of the effective reaction cavities in a droplet-type detection method. Therefore, the dynamic range of chip-based digital PCR is narrow relative to droplet-based PCR. The droplet PCR detection method disperses the sample into water-in-oil reaction units, and then performs real-time or end-point fluorescence analysis on each reaction unit.

Digital PCR (digital PCR, dPCR) is a newly developed third-generation PCR technology, mainly comprising large-scale integrated microfluidic chip digital PCR (cd PCR) and droplet digital PCR (ddPCR), wherein the two methods are to randomly distribute a DNA sample to be detected to form micro-reaction droplets before PCR amplification, each micro-reaction droplet contains either no nucleic acid target molecule to be detected or at least one nucleic acid target molecule to be detected, and each micro-reaction droplet is an independent PCR reactor. After PCR amplification, the detector detects each micro-reaction liquid drop, the liquid drop is judged to be '1' if the liquid drop has a fluorescence signal, the liquid drop is judged to be '0' if the liquid drop does not have the fluorescence signal, and finally the copy number or the concentration of the target sequence in the sample to be detected is directly calculated according to the Poisson distribution principle and the proportion of positive microdroplets.

Human adenoviruses (HAdV) are a family of viruses that contains over 60 serotypes, which are further grouped into 7 subgroups from a to G. It has a double-stranded DNA genome of about 35kb surrounded by an nonenveloped icosahedron, which has a fiber at each of the 12 vertices. The spike protein is non-covalently linked to the icosahedron by a pentameric polypeptide called the penton base.

According to previous reports, respiratory tract adenovirus infection in China is mainly seen in B1 group and B2 group of common respiratory tract adenovirus. Etiology monitoring data show that the subgenus B HAdV-7 and HAdV-3 are the main adenovirus types causing respiratory infectious diseases in China. The incidence of group respiratory tract adenovirus infection in the military is high, and the work and training of soldiers are influenced. Reports show that the group B adenovirus is the most common respiratory adenovirus of our army. Adenoviridae are also important causes of febrile illnesses in young children, most often causing upper respiratory syndromes such as pharyngitis or rhinocatarrh, but also pneumonia. In rare cases, adenoviruses can cause diseases of the digestive system, eye, urogenital system and nervous system.

Therefore, the subject group carries out sequence alignment on the H genes of the adenoviruses, and the H genes of the adenoviruses of the B1 group and the B2 group are found to have great difference, so 2 primer probes are respectively designed aiming at the H genes of the two groups of adenoviruses, and 3 groups of primer probes and 4 groups of primer probes are respectively designed for further screening.

Disclosure of Invention

Method

In one aspect, the invention provides a method for detecting group B1 adenoviruses, comprising performing PCR using an advh _ f3 upstream primer having the sequence shown in SEQ ID NO. 1 and an advh _ r3 downstream primer having the sequence shown in SEQ ID NO. 2.

Preferably, the detection includes identification, screening, typing, and the like.

Preferably, the adenovirus of group B1 includes, but is not limited to, adenovirus type 3 and adenovirus type 7.

Preferably, the method is for non-diagnostic purposes.

Preferably, the method further comprises using an advh _ p3 probe with the sequence shown in SEQ ID NO. 3 to embody the PCR result.

Preferably, both ends of the advh _ p3 probe are connected with a quencher and/or a fluorophore.

Preferably, the 5' end of the advh _ p3 probe is connected with a first fluorescent group.

Preferably, the 3' end of the advh _ p3 probe is connected with a first quenching group.

Preferably, the fluorophores (first fluorophore, second fluorophore) described herein include, but are not limited to, VIC, Texas Red, NED, PET, FAM (carboxyfluorescein, Green fluorescence), FITC (Fluorescein isothiocyanate), TET (Tetrachloro-6-carboxyfluorescein, Tetrachlorofluorescein), HEX (Hexachloro-6-methylfluorescein, Hexachloro Fluorescein), JOE (2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein), Rhodamine (Rhodamine dyes such as R110, TAMRA, Texas Red, etc.), ROX, Alexafluor dyes, ATTO dyes, DyLight dyes, cyanine dyes (e.g., Cy2, 3, Cy3.5, Cy b, Cy 36, Cy5, Cy 2.5, Cy 35, S3, Cy3.5, Cy b, Cy 36, Cy 35, Cy7, S5, S7, S5, S3, S3, S3, S.

Preferably, the Quencher group described herein (the first Quencher group, the second Quencher group) includes, but is not limited to, Tamra NHS, Dabcyl Quencher-3 ', Atto 540Q, Atto 575Q, Atto 612Q, BHQ-0(Black Hole Quencher 0, 3 '), BHQ-1(Black Hole Quencher 1, 3 '), BHQ-2(Black Hole Quencher 2, 3 '), BHQ-3(Black Hole Quencher 3, 3 '), BHQ-1(Black Hole Quencher-1, 5 '), Q-2(Black Hole Quencher-2, 5 '), BHQ-3(Black Hole Quencher-3, 5 '), BBQ-650NHS (Black Berry Quencher 650NHS), 3 ' -BBQ-650(Black Berry 650 BBQ 650), BHQ-650-BHQ-650 (Black Hole Quencher 650-BHQ-1, BHQ-3, 5 '), BHQ-1-BHQ-1 (Black Hole Quencher 650-BHQ-3, BHQ-3-BHQ-3, 5 '), BHQ-1 (BHQ-2, 3 '), BHQ-2, 3-BHQ-650 (BHQ-B-650-B-2, BHQ-650-B-2, BHQ-650-2, BHQ-3 ', BHQ-3-650-2, BHQ-650-2, BHQ-650-2, BHQ-650-3, BHQ-650-3-650-2, BHQ-B-3, BHQ-650-3-650-B-650, BHQ-2, BHQ-650, BHQ-2, BHQ-3, BHQ-2, BHQ-650, BHQ-3, BHQ-B-2, BHQ-B-650, BHQ-B-3, BHQ-B-650, BHQ-3, BHQ-B-3, BHQ-650, BHQ-3, BHQ-B-650, BHQ-3, BHQ-650, BHQ-B-2, BHQ-3, BHQ-B-2, BHQ-3, BHQ, BHQ-1-dT (Black Hole Quencher 1dT), BHQ-2-dT (Black Hole Quencher 2dT), Dabcyl Quencher deoxythymidine dT, MGB 3 'CDPI 3, MGB-5' CDPI3, TAMRA-3 '(carboxyethylene diamine), Dabcyl-5', Tamra-dT carboxyethylene diamine-dT).

Preferably, the 5' end of the advh _ p3 probe is connected with FAM fluorescent group.

Preferably, the 3' end of the advh _ p3 probe is connected with a BHQ1 quenching group.

Preferably, the method further comprises the step of performing PCR on the internal reference gene.

Preferably, the reference gene comprises RPP 30.

Preferably, the reference gene is RPP 30.

Preferably, the PCR of the reference gene comprises PCR using an upstream primer of hadvh55_ f4 with the sequence shown in SEQ ID NO. 4 and a downstream primer of hadvh55_ r4 with the sequence shown in SEQ ID NO. 5.

Preferably, the PCR of the reference gene comprises embodying the PCR result using a hadvh55_ p4 probe having the sequence shown in SEQ ID NO 6.

Preferably, the two ends of the hadvh55_ p4 probe are connected with a quenching group and/or a fluorescent group.

Preferably, the 5' end of the hadvh55_ p4 probe is connected with a second fluorescent group.

Preferably, the 3' end of the hadvh55_ p4 probe is connected with a second quenching group.

Preferably, the second fluorophore is the same or different from the first fluorophore.

Preferably, the second fluorophore is not the same as the first fluorophore.

Preferably, the second quencher is the same or different from the first quencher.

Preferably, the second quencher is the same as the first quencher.

Preferably, the 5' end of the hadvh55_ p4 probe is connected with a VIC fluorescent group.

Preferably, the 3' end of the hadvh55_ p4 probe is connected with a BHQ1 quenching group.

Preferably, the PCR comprises an amplification reaction based on the principle of PCR.

Preferably, the PCR is digital PCR (dpcr).

Preferably, the digital PCR includes a droplet-type digital PCR (ddPCR) and a chip-type digital PCR (chamberbasededigital PCR).

Preferably, the digital PCR is ddPCR.

Preferably, the method further comprises the step of extracting and/or processing the sample.

Preferably, the sample comprises a sample taken from a human or from the environment.

Preferably, the collecting of the sample from the human body comprises serum, whole blood, stool, throat swab, nasal discharge, saliva, sweat, hair, buccal cells, pus, cerebrospinal fluid, semen.

Preferably, the sample from the environment comprises a bacterial culture, bacterial colony, viral suspension, environmental concentrate, foodstuff, raw material, water sample, or water concentrate.

Preferably, the method further comprises the step of reading the results (values, images) and/or quantitative calculations.

Preferably, modified nucleotides may be included in the primers or probes of the present invention.

Preferably, the modifications include phosphate backbone modifications, base modifications, ribose modifications.

Preferably, the phosphate backbone modification may be a thio modification, meaning that the non-bridging oxygen atom in the phosphodiester linkage in the nucleotide fragment is replaced by a sulfur atom.

Preferably, the modification of the phosphate backbone may also be achieved by forming other types of phosphate linkages including, but not limited to, methyl phosphate, selenophosphate, boronophosphate, and dithiophosphate.

Preferably, the ribose modification may be a modification of the 2' position of the pentose sugar. Modifications at the 2' position of the pentose include, but are not limited to, methoxy, methoxyethoxy, propenyloxy, and fluoro modifications.

Preferably, the base modification mainly refers to the inclusion of rare bases in the single-stranded deoxynucleotide, and the rare bases refer to some bases except A, G, C, U, including Dihydrouracil (DHU), pseudouracil and methylated purine (mG, mA).

Preferably, the kit may further comprise reagents and/or instruments for detecting other target nucleic acids;

preferably, the other target nucleic acids include animal genomes, plant genomes, and microorganisms;

preferably, the microorganisms include prions, parasites (protozoa, worms, medical insects), fungi, bacteria, spirochetes, mycoplasmas, rickettsiae, chlamydia and viruses;

preferably, the virus includes adenovirus, hepatitis virus, influenza virus, varicella virus, herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), rinderpest virus, respiratory syncytial virus, cytomegalovirus, echinovirus, arbovirus, hantavirus, mumps virus, measles virus, rubella virus, human immunodeficiency virus type I (HIV-1), human immunodeficiency virus type II (HIV-2), any togavirus virus (e.g., dengue virus), alphavirus, flavivirus, coronavirus, rabies virus, green monkey virus, ebola virus, parainfluenza virus, orthomyxovirus, arenavirus, human T cell leukemia virus type I, human T cell leukemia virus type II, simian immunodeficiency virus, lentivirus, epstein-barr virus, Human herpesvirus, herpes similis virus, poxvirus, etc.;

product(s)

In another aspect, the invention provides a pair of primers for detecting adenovirus group B1, wherein the primers comprise an advh _ f3 upstream primer shown as SEQ ID NO. 1 and an advh _ r3 downstream primer shown as SEQ ID NO. 2.

Preferably, the primer is composed of an advh _ f3 upstream primer shown in SEQ ID NO. 1 and an advh _ r3 downstream primer shown in SEQ ID NO. 2.

In another aspect, the invention provides a set of primer probe combinations for detecting adenoviruses of group B1, wherein the primers comprise the aforementioned advh _ f3 upstream primer and the aforementioned advh _ r3 downstream primer, and the probes are the aforementioned advh _ p3 probes.

In another aspect, the invention provides a kit for detecting adenovirus group B1, wherein the kit comprises the primer for detecting adenovirus group B1 or the primer probe combination for detecting adenovirus group B1.

Preferably, the kit further comprises the primer and the primer probe combination used for carrying out PCR on the internal reference gene.

Preferably, the kit further comprises a DNA polymerase.

Preferably, the kit further comprises reagents used for preparing the PCR reaction system.

Preferably, the kit further comprises the instruments necessary to carry out the aforementioned PCR reactions.

Applications of

In another aspect, the invention provides an application of the primer, the primer probe combination or the kit in detecting B1 group adenoviruses.

In another aspect, the invention provides an application of the primer, the primer probe combination or the kit in preparation of a product for detecting B1 group adenovirus.

Drawings

FIG. 1 is a diagram of the digital PCR detection visualization result of the primer probe combination of group 1 for reference gene detection.

FIG. 2 is a diagram of the visualized results of digital PCR detection of the primer probe combination of group 2 for reference gene detection.

FIG. 3 is a diagram of the digital PCR detection visualization result of the 3 rd group primer probe combination for reference gene detection.

FIG. 4 is a diagram showing the digital PCR detection visualization results of detecting adenovirus type 3 and healthy human throat swab mixtures by using B1 group adenovirus primer probes and internal reference gene primer probe mixtures.

FIG. 5 is a diagram showing the results of digital PCR detection using the B1 group adenovirus primer probe and reference gene primer probe mixture for detecting adenovirus type 7 and healthy human throat swab mixture.

FIG. 6 is a diagram showing the results of digital PCR detection of adenovirus type 11 and throat swab mixtures of healthy humans using the B1 group adenovirus primer probe and reference gene primer probe mixture.

FIG. 7 is a graph showing the results of digital PCR detection of adenovirus type 14 and throat swab mixtures of healthy humans using the B1 group adenovirus primer probe and reference gene primer probe mixture.

FIG. 8 is a diagram showing the results of digital PCR detection of adenovirus type 55 and throat swab mixtures of healthy humans using the B1 group adenovirus primer probe and reference gene primer probe mixture.

FIG. 9 is a diagram of the visualization result of digital PCR detection of type 3 and type 55 mixed samples using B1 group adenovirus primer probes and reference gene primer probe mixture.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 screening of PCR sites and design of primers and probes

Subject group carries out sequence comparison on H genes of B1 group adenoviruses, finds that the specificity of B1 group adenoviruses H genes is higher, and further selects different positions on the H genes as targets to design three groups of primer probes, wherein the sequences of the primer probes are shown in Table 1:

TABLE 1 primer and probe sequences for detection of group B1 adenoviruses

qPCR detection was performed using adenovirus type 3 as template and the above three sets of primers, and detection was performed using One Step PrimeScript from takara^TMRT-PCR Kit (Perfect Real Time) (Code No. RR064) Kit, reaction system total 25 ul, wherein 2 Xone Step RT-PCR Buffer 12.5 ul, TaKaRa Ex TaqHs 0.5 ul, Rox Reference Dye II 0.5 ul, primer probe mixture 3 ul, RNase Free dH₂O 6.5μl，DNA2μl。

The reaction conditions were 95 ℃ for 10s, 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles (fluorescence signal was collected). The experiment was performed on an ABI7500 instrument.

From the data, Ct value results are shown as:

group 1: >40

Group 2: 25.4

Group 3: 17.4

The same primer probe combination and experimental method are used for detecting adenovirus type 7, and the Ct value result is shown as follows:

group 1: 17.5

Group 2: 22.8

Group 3: 12.2

Combining the above results, the ct value of group 3 was the lowest, and then the primer of group 3 was compared with the primer probe combination sold in Meining kang City (manufacturer: Nanjing Meining kang City Biotech Co., Ltd.; product: respiratory adenovirus DNA detection kit; product number: BP 0211-32):

and (3) comparing the detection results of the adenovirus type 3: the ct value of the primer is 17.46, and the ct value of the adenovirus detection reagent sold in Meining Kangcheng is 18.64;

comparison of the detection results for adenovirus type 7: the ct value of the primer is 12.2, and the ct value detected by adenovirus sold in Meining Kangcheng is 11.9.

The above experiments show that the primers and probes of the present invention can rapidly detect the presence of B1 group (type 3, type 7, etc.) adenoviruses in a sample.

Example 2 screening of reference Gene and primer Probe therefor

Subject groups used the respiratory tract housekeeping gene RPP30 as an internal reference gene, and 3 primer controls were set, as shown in table 2:

TABLE 2 primer and probe sequences for detection of reference gene of RPP30

Throat swab samples from subjects maintained in the subject group were selected for digital PCR testing using an TARGETING ONE system including a droplet generator and a droplet reader (neoyobium) a300 Fast Thermal Cycler rapid gradient PCR instrument (LongGene lang).

Reaction system: premix 15 μ l [ primer probe mixture ]: 500nM primer ADV7, 200nM probe, 600nM primer RPP30, 300nM probe (final concentration in PCR system), 3. mu.l ADV7+ 3. mu.l RPP30, 7. mu.l l H₂O, 2. mu.l template.

The amplification conditions were: 600s at 95 ℃, 30s at 94 ℃, 60s at 55 ℃, 42 cycles at 12 ℃ for 300s, and the variable temperature speed is set to be 1.5 degrees/second.

The sample copy number results of the digital PCR assay are shown in table 3:

TABLE 3.3 copy number of primer-probe combinations tested

Sample numbering	1	2	3
				Sample remark information	z2，rpp30-1	z2，rpp30-2	z2，rpp30-3
Copy number (VIC)	357.3	467.6	374.6

Meanwhile, as shown in FIGS. 1-3, the visualized digital PCR detection chart is characterized in that green (light color, right side) data points are positive points, the data points of the third group of primer probe combination RPP30-3 are the most dense, the data points are most obviously distinguished from the black data points of the control, and the detection effect is the best.

The above data indicate that the probe primer for internal reference gene screening can stably detect the internal reference RPP30 and can play a role in indicating the detection effect, so that the primer probe combination screened in this example is continuously used for the next experiment.

Example 3 and B1 results of detecting AdV3, AdV7, AdV11, AdV14 and AdV55 type AdVs

The detection effect of the B1 group adenovirus detection primer probe selected in example 1 and the reference gene primer probe selected in example 2 in digital PCR detection is verified. Wherein the healthy human pharynx swab extract mainly provides an internal reference gene for a sample to be detected.

The instrument comprises the following steps: xin Yi TD-1 micro-drop digital PCR system, A300 Fast Thermal Cycler Fast gradient PCR instrument (LongGene Langi).

Amplification conditions: 95 ℃ 600s, 94 ℃ 30s, 55 ℃ 60s, 42 cycles, 12 ℃ 300s attention: the ramp rate was set at 1.5 degrees/second. The results are as follows:

the results are as follows:

FIG. 4 shows the digital PCR detection results of the adenovirus type 3 and healthy human throat swab mixtures using the B1 group adenovirus primer probe and reference gene primer probe mixture, and the results of 3 replicates are shown. The copy numbers of the type 3 adenovirus detection of the three detections are respectively as follows: 33153.4, 30616.5, and 34048.7; the copy numbers of the internal reference genes detected for three times are respectively as follows: 286.8, 250.7 and 286.1.

FIG. 5 shows the result of digital PCR detection of adenovirus type 7 and throat swab mixtures of healthy humans using the B1 group adenovirus primer probe and reference gene primer probe mixture, and the result of 3 replicates. Wherein the copy number of the type 7 adenovirus is respectively as follows: 3874.8, 3725.6, and 3410.9; and the reference gene copy number is: 281.8, 308, and 295.

The results show that the relevant primers and probes have good and stable detection effect in digital PCR detection.

FIG. 6 is a digital PCR detection result chart of B1 group adenovirus primer probe and reference gene primer probe mixture for detecting 11 type adenovirus and healthy human throat swab extract mixed samples. Wherein the detected copy number of the adenovirus is 0, and the detected copy number of the internal reference gene is 312.2, 292.8 and 285.5.

FIG. 7 is a graph showing the results of digital PCR detection of mixed samples of adenovirus type 14 and healthy human throat swab extracts by using B1 group adenovirus primer probes and internal reference gene primer probe mixtures, and the results of 3 replicates. Wherein the adenovirus detection copy number is 0, and the internal reference gene detection copy numbers are 274.1, 312.1 and 231.2.

FIG. 8 is a graph showing the results of digital PCR detection of mixed samples of adenovirus type 55 and healthy human throat swab extracts by using B1 group adenovirus primer probes and internal reference gene primer probe mixtures, and the results of 3 replicates. Wherein the adenovirus detection copy number is 0, and the internal reference gene detection copy numbers are 235.3, 273.9 and 267.7.

The results show that the mixture of the adenovirus primer probe and the internal reference gene primer probe in the B1 group has good detection specificity.

FIG. 9 shows the results of the detection of mixed samples of group B1 (type 3) and group B2 (type 55), indicating that only type 3 adenovirus, but not type 55 adenovirus, was detected; the primer and the probe of the invention have good specificity.

Example 4 verification of B1 group adenovirus primer probes and Meining Kangcheng sale adenovirus designed by the invention by qPCR Advantages of the detection kit in patient detection

The qPCR detection was performed using 5 samples of throat swabs of adenovirus type 7 infected patients, using the B1 group adenovirus primer probes and the Meining Kangcheng sold adenovirus detection kit designed according to the present invention, and the results are shown in Table 4 below:

TABLE 4 sample name and test results

The results of this example show that the primer probe designed by the present invention is slightly better than the adenovirus primer probe sold in Meining kang City in the qPCR detection of the clinical sample. Among them, in the detection of SAMPLE2 and SAMPLE4, the ct value detected by the Meiningcong reagent can be judged as a gray zone detection value or a value close to the gray zone detection value, while the primer probe combination designed by the invention is about 33, and can be directly judged as positive, which shows that the primer probe combination in the invention has certain advantages in the SAMPLE detection at the critical value.

Example 5 determination of minimum detection Limit

Throat swab samples of 60 healthy persons were examined using the primer probe set B1 plus another primer probe set of this study (primer probe set B2, shown in SEQ ID NO:7-9 in sequence) plus the primer probe set for the reference gene, and the results are shown in Table 5.

The lowest detection limit of 5copies/test was calculated (calculation method shown in Table 6, reference (Milbury et al 2014)). The lowest detection limit of the primer probes of the B1 group when used alone is not higher than the lowest detection limit after mixing. Therefore, the respective minimum detection limits of the primer probe combinations of the B1 group/the reference gene primer probe combination/the B2 group are 5copies/test or less than 5 copies/test.

The B1 group primer probe provided by the invention has good specificity in detection and is not influenced by the internal reference probe primer or the B2 group primer probe.

TABLE 5.60 digital PCR assay data for healthy persons

TABLE 6 calculation of minimum detection limit

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Claims

1. A method for identifying group B1 adenoviruses in adenoviruses for non-diagnostic purposes, the method comprising performing digital PCR using an advh _ f3 upstream primer having the sequence shown in SEQ ID NO. 1, an advh _ r3 downstream primer having the sequence shown in SEQ ID NO. 2, and an advh _ p3 probe having the sequence shown in SEQ ID NO. 3,

the B1 group adenoviruses are type 3 adenoviruses and type 7 adenoviruses.

2. The method of claim 1, wherein the advh _ p3 probe is linked at both ends to a quencher and a fluorophore.

3. The method of claim 2, wherein the 5' end of the advh _ p3 probe is linked to a FAM fluorophore.

4. The method of claim 2, wherein the advh _ p3 probe is linked at its 3' end to a BHQ1 quencher.

5. The method of claim 1, further comprising the step of performing digital PCR on an internal reference gene, said internal reference gene comprising RPP 30.

6. The method of claim 5, wherein performing PCR on the reference gene comprises performing digital PCR using a hadvh55_ f4 upstream primer having the sequence shown in SEQ ID NO. 4, a hadvh55_ r4 downstream primer having the sequence shown in SEQ ID NO. 5, and a hadvh55_ p4 probe having the sequence shown in SEQ ID NO. 6.

7. The method of claim 6, wherein the hadvh55_ p4 probe has a quencher and a fluorophore attached to each end.

8. The method of claim 7, wherein the hadvh55_ p4 probe is 5' linked to a VIC fluorophore.

9. The method of claim 7, wherein the hadvh55_ p4 probe is 3' linked to a BHQ1 quencher.

10. The method of claim 1, wherein the digital PCR is a droplet digital PCR.

11. The method of claim 1, further comprising the step of extracting the sample.

12. The method of claim 11, wherein the sample comprises a sample from an environment.

13. The method of claim 12, wherein the sample from the environment comprises a bacterial culture, a viral suspension, an environmental concentrate, a foodstuff, a water sample, or an aqueous concentrate.

14. A group of primer probe combinations for detecting B1 group adenoviruses comprises an advh _ f3 upstream primer with a sequence shown as SEQ ID NO. 1, an advh _ r3 downstream primer with a sequence shown as SEQ ID NO. 2 and an advh _ p3 probe with a sequence shown as SEQ ID NO. 3,

the B1 group adenoviruses are type 3 adenoviruses and type 7 adenoviruses.

15. A kit for detecting adenovirus group B1, the kit comprises the primer probe combination for detecting adenovirus group B1 of claim 14,

the B1 group adenoviruses are type 3 adenoviruses and type 7 adenoviruses.

16. The kit of claim 15, further comprising a hadvh55_ f4 upstream primer having the sequence shown in SEQ ID No. 4 and a hadvh55_ r4 downstream primer having the sequence shown in SEQ ID No. 5.

17. The kit of claim 16, further comprising a hadvh55_ p4 probe having the sequence shown in SEQ ID No. 6.

18. The application of the primer probe combination for detecting the adenovirus in the B1 group in claim 14 or the kit for detecting the adenovirus in the B1 group in claim 15 in preparing products for identifying the adenovirus in the B1 group,

the B1 group adenoviruses are type 3 adenoviruses and type 7 adenoviruses.